An important characteristic of cellular senescence is the loss of proliferative capacity, during which cells exhibit morphological changes and alterations in gene expression. One function of cellular senescence is to suppress the initiationof tumors. Gene expressionin eukaryotic cells is regulated to a large extent by post-transcriptional modification of pre-mRNA, which includes polyadenylation, cleavage and alternative splicing. The relevance of post-transcriptional processing has not been investigated in cellular aging. We have identified alterations in the levels and activities of proteins which bind to heterogeneous nuclear RNA (hnRNA) in senescent fibroblasts; these are the hnRNP A1 and A2 proteins, and both are functional in the biogenesis and stability of mature mRNA. The consequences of altered activities of RNA binding proteins could potentially produce several effects, such as reduced mRNA, defective proteins, and inefficient translation. All are possible mechanisms to induce cellular senescence. Through the course of our SCORE grant, we have found that overexpression of either hnRNP A1 or A2 results in the increased expression of two mRNA isoformsof the INK4a locus, p14 ARFand p16 INK4a . Both proteins are growth inhibitors and are important cell cycle modulatorsfor replicative senescence and immortalization.Our findings are the first to show that post-transcriptional processing may exert a significant effect on the maintenance of the senescent phenotype. Our hypothesis is that alterations in post-transcriptional processing has an important role in affecting gene expression during cellular senescence. The goals of this proposal are to investigate the age-related roles of hnRNP A1 and A2 binding proteins. We will further characterize their biochemical activities and biological functions during cellular senescence. We will investigate mechanisms whereby they modulate the expression of p14 ARF and p16 INK4a mRNA isoforms and subsequent effects on p14 ARFand p16 INK4a pathways. One long term goal of the studies outlined in this application is to characterize the expression of genes modulated by hnRNP A1 and A2 during senescence that have been identified by cDNA microarray analysis. The studies proposed will assess the contribution post-transcriptional processing has in modulating senescence-related gene expression.